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Hyperglycemia Enhances Extracellular Glutamate Accumulation in Rats Subjected to
Forebrain Ischemia
Ping-An Li, Ashfaq Shuaib, Hiro Miyashita, Qing-Ping He and Bo K. Siesjö
Stroke. 2000;31:183-192
doi: 10.1161/01.STR.31.1.183
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Hyperglycemia Enhances Extracellular Glutamate
Accumulation in Rats Subjected to Forebrain Ischemia
Ping-An Li, MD, PhD; Ashfaq Shuaib, MD, FRCPC;
Hiro Miyashita, MS; Qing-Ping He, MS; Bo K. Siesjö, MD, PhD
Background and Purpose—An increase in serum glucose at the time of acute ischemia has been shown to adversely affect
prognosis. The mechanisms for the hyperglycemia-exacerbated damage are not fully understood. The objective of this
study was to determine whether hyperglycemia leads to enhanced accumulation of extracellular concentrations of
excitatory amino acids and whether such increases correlate with the histopathological outcome.
Methods—Rats fasted overnight were infused with either glucose or saline 45 minutes before the induction of 15 minutes
of forebrain ischemia. Extracellular glutamate, glutamine, glycine, taurine, alanine, and serine concentrations were
measured before, during, and after ischemia in both the hippocampus and the neocortex in both control and
hyperglycemic animals. The histopathological outcome was evaluated by light microscopy.
Results—There was a significant increase in extracellular glutamate levels in the hippocampus and cerebral cortex in
normoglycemic ischemic animals. The increase in glutamate levels in the cerebral cortex, but not in the hippocampus,
was significantly higher in hyperglycemic animals than in controls. Correspondingly, exaggerated neuronal damage was
observed in neocortical regions in hyperglycemic animals.
Conclusions—The present results demonstrate that, at least in the neocortex, preischemic hyperglycemia enhances the
accumulation of extracellular glutamate during ischemia, providing a tentative explanation for why neuronal damage is
exaggerated. (Stroke. 2000;31:183-192.)
Key Words: cerebral ischemia n excitatory amino acids n glutamates n hyperglycemia n pathology n rats
I
t has been known for 2 decades that preischemic hyperglycemia aggravates brain damage due to transient global
or forebrain ischemia in experimental animals. Clinical studies on diabetic and hyperglycemic patients support the notion
that raised plasma glucose concentrations augment brain
lesions associated with stroke.1 The aggravation in global
ischemia has the following features: (1) the neuronal necrosis
evolves more rapidly, (2) additional structures are recruited,
(3) selective neuronal necrosis is transformed into pannecrosis, and (4) postischemic seizures develop and then end in
fatal status epilepticus (for reviews, see References 2 and 3).
The mechanisms involved have not been adequately defined. Hyperglycemia probably acts by enhancing accumulation of lactate plus H1, thereby exaggerating the reduction of
intracellular and extracellular pH (pHi and pHe, respectively).
In support of this notion are the results reported by Smith et
al,4 (1986) showing that hyperglycemic subjects have more
profound decreases in pHi and pHe during ischemia, and those
of Katsura et al,5 (1992), demonstrating an almost linear
correlation between the increase in lactate during ischemia
and the fall in pHi and pHe. Furthermore, Li et al6,7 (1994,
See Editorial Comment, page 191
1995) reported a very narrow threshold of plasma glucose
concentration (10 to 14 mmol/L) and of pHe (6.3 to 6.5),
above and below which brain damage was exaggerated and
postischemic seizures were triggered, supporting the notion
of a critical pH range.
It has been established that excitatory amino acids (EAAs),
notably glutamate, play a pivotal role in neuronal death.8 –10
Recent studies demonstrate that mitochondria are a primary
target of glutamate toxicity.11 Glutamate-mediated massive
influx of calcium triggers opening of a mitochondrial permeability transition pore, which may contribute to cell death.
The possibility that hyperglycemia exaggerates brain damage
by increasing the efflux of glutamate has not been established. In the present study we used in vivo microdialysis
coupled with high-performance liquid chromatography
(HPLC) techniques to detect the pattern of changes in
extracellular glutamate, glutamine, glycine, taurine, alanine,
and serine concentrations in brain tissue of rats subjected to
15 minutes of forebrain ischemia. We also evaluated the
Received May 4, 1999; final revision received September 7, 1999; accepted October 12, 1999.
From the Saskatchewan Stroke Research Centre, University of Saskatchewan, Saskatoon, Canada (P-A.L., A.S., H.M., Q-P.H.); Department of
Medicine (Neurology), University of Alberta, Edmonton, Canada (A.S.); and Center for the Study of Neurological Disease, Queen’s Neuroscience
Institute, Queen’s Medical Center, Honolulu, Hawaii (P-A.L., Q-P.H., B.K.S.).
Reprint requests to Ashfaq Shuaib, Department of Medicine (Neurology), 2E.13 Walter Mackenzie Health Science Centre, 8440-112th St, Edmonton,
AB T6G 2B7, Canada. E-mail [email protected]
© 2000 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
183
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January 2000
pathological outcome in separate groups of animals. The
hypothesis tested was whether hyperglycemia enhances the
rise in extracellular glutamate concentration and whether it
correlates with exaggerated tissue damage.
Materials and Methods
Male Wistar rats of a specific-pathogen-free strain (Charles River
Animals Facility, Quebec, Canada), weighing 300 to 390 g, were
used in the present experiments. All animal use procedures were in
strict accordance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals and were approved by the
Animal Ethics Committee of the University of Saskatchewan. All
efforts were made to minimize animal suffering, to reduce the
number of animals used, and to use alternatives to in vivo techniques,
if available.
Implantation of Guide Cannulas
nated by reinfusion of the shed blood and removal of the carotid
clamps.
Experimental Groups
Twenty animals were divided into 3 groups for microdialysis studies.
Experimental groups included 16 animals subjected to 15 minutes of
transient forebrain ischemia with preischemic saline or glucose
infusion (normoglycemia and hyperglycemia; n58 in each). Four
sham-operated rats served as control (n54). In another series, 20
animals were used for the evaluation of histopathological outcome in
normoglycemic and hyperglycemic animals. Hyperglycemic animals
were infused with a 25% glucose solution (2.5 to 3.0 mL/h) for 45
minutes before ischemia to yield a plasma glucose concentration of
'370 g/100 mL, while normoglycemic rats were infused with the
same amount of normal saline solution.
In Vivo Microdialysis Procedure
Five to 7 days before microdialysis experiments, the animals were
anesthetized with Equitensin (a mixture of pentobarbital, propylene
glycol, ethanol, MgSO4, and chloral hydrate). The hair on the skull
was shaved, and the skin was treated with an antiseptic. The animals
were placed in a stereotaxic frame (David Kopf) for positioning of
the guide cannula of the microdialysis probes in the hippocampus
and cortex. The skull was exposed and treated with 3% hydrogen
peroxide. Three metal screws were firmly connected to the skull for
fixation of a guide cannula on the skull. Two burr holes were made
in the cranium. One was made overlying the left dorsal hippocampus
3.8 mm posterior to bregma, 2.5 mm lateral to midline, and 1.5 mm
ventral to dura. The other was drilled over the right parietal cortex
3.0 mm posterior to bregma, 5.0 mm lateral to midline, and 2.0 mm
ventral to dura. The probe length was 2 mm longer than the guide
cannula, resulting in a final depth, after inserting the probe, of 3.5
and 4.0 mm in the hippocampus and neocortex, respectively. After
insertion, the guide cannulas were fixed to the skull by placing dental
acrylic around the cannula. The scalp was then closed surgically, and
the animal was returned to its home cage, being individually housed.
After the operation was finished, a microdialysis probe with a
2.0-mm membrane length (CMA 12, Carnegie Medicine AB) was
inserted through the preimplanted guide cannula into the left dorsal
hippocampus and the right parietal cortex. The probe was perfused
with a modified Ringer’s solution (mmol: NaCl 145, KCl 2.7, CaCl2
1.2, MgCl2 1.2, pH 7.4) at a flow rate of 2 mL/min by means of a
microinfusion pump (CMA/microdialysis, 100 microinjection pump,
Carnegie Medicine AB). Transient increases in amino acids and
other neurotransmitters from probe insertion damage were avoided
by a 90-minute stabilization period. Microdialysis sampling was
performed at 15-minute intervals for 180 minutes. Three samples
were collected before ischemia, 1 sample during ischemia, and 8
samples during the reperfusion period. All samples were collected on
an ice bath and then stored at 220°C until analysis. At the end of
each experiment, the microdialysis probes were removed, and the
recovery rate for each probe was determined with the use of a 100
pg/10 mL standard solution in vitro. The recovery rate of interstitial
amino acids was 8% to 13%. In 6 animals, the location of the
microdialysis probe was verified on brain sections after the
experiment.
Operative Procedures
Detection and Quantification of EAAs
The animals were fasted overnight before surgery with free access to
water. Anesthesia was induced by inhalation of 3.5% halothane in a
mixture of N2O and O2 (60:40) and was maintained with 1.0% to
1.5% halothane in N2O and O2 (60:40) during the operation. A
midline incision of the neck was performed. Both common carotid
arteries were isolated, and loose threads were placed for subsequent
occlusion of the arteries. A flexible silicone catheter was inserted
into the vena cava via the right jugular vein for withdrawal of blood.
One tail vein and the artery were cannulated for infusion of glucose,
injection of drugs, and monitoring of blood gases, pH, and blood
pressure. A rectal thermometer (Thermocouple Thermometer, Barnant Company) was inserted, and another thermometer was placed
subcutaneously on the skull to monitor the body and head temperatures, which were maintained as close to 37°C as possible by lamp
heating during the whole process. Electroencephalographic (EEG)
needles were inserted into the temporal muscles on the skull. The
animals were heparinized with 30 IU/kg of heparin. Blood gases
were measured with a micro sample blood gas monitor (ABL 300,
Radiometer). Blood pressure was monitored and recorded on a Grass
model 7D polygraph (Grass Instrument Co). Blood glucose concentration was determined by an Ames Glucometer II/Glucostix system
(Miles Canada Inc).
Analysis of extracellular concentrations of glutamate, glutamine,
glycine, taurine, alanine, and serine in the microdialysate was
performed by HPLC (model 510 Liquid Pump equipped with a
10-mm filter; Millipore Corporation, Waters Chromatography Division) with electrochemical detection after precolumn derivatization
by methods described previously in detail.13 A 5.0-mm Delta-Pak
Cartridge filter was installed between the pump and a 715 Ultra Wisp
Sample Processor (Millipore Corporation). A 4.00-mm C18 column
preceded a reverse-phase Nova-Pak steel column (Waters)
(3.93150 mm) (4.0 particle size), which in turn, was connected to a
model 460 High Sensitivity Waters Electrochemical Detector.
Precolumn derivatization of amino acids with o-phthaldialdehyde/
b-mercaptoethanol was done before electrochemical detection. Using the 715 Ultra Wisp Sample Processor (Millipore Corporation),
we employed a fully automated system for the derivatization
procedure to minimize the degradation of amino acids. This sample
processor dispenses and mixes the reagent thoroughly into the
microdialysis perfusate in a 200-mL loop. The injection was made
precisely 2 minutes after the reaction time. Amino acid concentrations were measured and stored in the computer for future analysis
with the Baseline 810 Chromatography Work Station Processing
System (Waters). The mobile phase consisted of 0.01 mol/L disodium hydrogen orthophosphate, 0.13 mmol ethylenediaminetetraacetic acid–sodium salt, and 20% methanol. External standards of 0.625,
1.25, 2.5, 25, 100, 300, and 1200 pg/10 mL were used for postexperiment probe calibration for all the amino acids.
Induction of Ischemia
Reversible forebrain ischemia of 15 minutes’ duration was induced
by a combination of bilateral carotid artery clamping and hypotension, the latter being achieved by withdrawal of blood through the
catheter inserted into the jugular vein.12 Blood pressure was maintained at 40 to 50 mm Hg during the ischemic period. The EEG was
monitored before, during, and after ischemia. A flat EEG was
considered to constitute the onset of ischemia. Ischemia was termi-
Quantification of Brain Damage
None of the normoglycemic animals subjected to 15 minutes of
forebrain ischemia at 37°C body and brain temperature developed
postischemic seizures, and all survived for 7 days, allowing conven-
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Li et al
Effect of Glucose on Excitatory Amino Acids
185
tional histopathological evaluation of brain damage. In hyperglycemic animals, however, survival was severely restricted. Thus, when
6 animals in a pilot study were allowed to wake up after the
ischemia, all developed seizures during the first 1 to 3 hours and
subsequently died in status epilepticus. For that reason, brain damage
in hyperglycemic animals was evaluated after only 3 hours of
reperfusion and was compared with that incurred in normoglycemic
animals.
For perfusion-fixation, animals were reanesthetized with 3%
halothane, tracheotomized, and artificially ventilated. The thorax
was opened, and a perfusion needle was transcardially inserted into
the ascending aorta. The brains were first rinsed with saline for 30
seconds and then perfusion-fixed with 4% formaldehyde buffered to
pH 7.35. The brains were cut coronally in 2- to 3-mm-thick slices,
dehydrated, embedded in paraffin, sectioned in 5 mm with a Reichert
sledge microtome, and then stained with acid fuchsin and celestine
blue, as described by Auer et al (1984).14 The sections were
examined in a blinded fashion at a magnification of 3400. Any
necrotic neurons of both hemispheres were counted in one coronal
section at the level of bregma 23.8 mm in the hippocampal CA1
sector, and the percentage of necrotic neurons was calculated by
dividing the number of necrotic neurons by the total number of
existing neurons at the identical brain section level in nonoperated
normal animals. Damage in the neocortex was too extensive to count
through the whole section in hyperglycemic animals. Therefore, the
damage was assessed by counting dead neurons in a strip, 400 mm
wide, through layers 1 to 6 at a site just between the midline and the
entorhinal fissure at the level of bregma 23.8 mm. The average total
number of neurons in this area was 450 per stripe in nonoperated
normal animals. The percentage of necrotic neurons was calculated.
Statistical Analysis
EAA concentrations are presented as mean6SEM. Paired t test (for
chronological data versus preischemic baseline) and unpaired t test
(for hyperglycemia versus normoglycemia at identical sampling
time) were applied for statistical analysis. All tests were 2-sided.
P,0.05 was considered statistically significant.
Results
Figure 1. Photomicrographs show neocortical damage after 3
hours of recovery in both normoglycemic and hyperglycemic
animals. Although the majority of neocortical neurons were morphologically intact in the normoglycemic rat, a few darkly
stained preacidophilic neurons were present in the center of the
field (A). In contrast, all neurons were necrotic in the hyperglycemic animal (B). In addition, brain edema was present in the
hyperglycemic animal. Bar550 mmol/L.
Physiological Parameters and Seizure Incidence
Blood glucose concentrations were 90.0614.4 and
372.6627.2 g/100 mL in normoglycemic and hyperglycemic
animals, respectively. Both head and body temperatures were
maintained to 37.260.2°C. Mean arterial blood pressure was
maintained at 120613 mm Hg. Ventilation was adjusted to
give an arterial pH of 7.460.2, PCO2 of 40.262.2 mm Hg, and
PO2 of 104618 mm Hg. There were no statistically significant
differences between normoglycemic and hyperglycemic
groups. EEG was continuously monitored during the entire
microdialysis periods, and no subclinical seizure activities
were observed in either normoglycemic or hyperglycemic
rats. In the series for histopathological study, no animal
developed postischemic clinical seizures in normoglycemic
subjects, while 4 of 6 showed seizure in hyperglycemic
animals. Pathological evaluation showed that the 2 seizurefree rats had the same extent of damage as rats with seizures.
Histopathological Outcome
Predictably, normoglycemic animals did not have any damage in the hippocampal CA1 sector after 3 hours of recovery,
and only a few scattered necrotic neurons were found in the
neocortex. When the recirculation was prolonged for 7 days,
the ischemia gave rise to nearly 100% damage in the CA1
sector and 30% damage in the neocortex. In the hyperglycemic animals, only a few necrotic neurons could be observed
in the CA1 sector (5% damage) after the 3-hour recovery
period. The small amount of CA1 damage in both normoglycemic and hyperglycemic animals after 3 hours of reperfusion
was probably due to the fact that such a short period of
reperfusion did not allow the CA1 damage to mature.15
Damage in the neocortex was dramatically exaggerated in
hyperglycemic rats (P,0.01 versus normoglycemic controls). Pannecrotic lesions were observed in 5 of 6 hyperglycemic animals; additionally, status spongiosus suggested
edema. Figure 1 contains representative microphotographs
showing neuronal damage after 3 hours of reperfusion in the
neocortical area in both normoglycemic and hyperglycemic
rats. Adverse effects of hyperglycemia on tissue damage
could also be observed in other brain areas. Thus, while only
mild to moderate damage was observed in the striatum and
thalamus in normoglycemic animals after 7 days of recovery,
pannecrosis occurred in these structures of hyperglycemic
animals (data not shown) as early as after 3 hours of recovery.
In addition, damage to the cingulate cortex and the substantia
nigra was not observed unless hyperglycemia was instituted
(data not shown).
Extracellular Amino Acids
Three microdialysis samples were collected at 15-minute
intervals to obtain a stable preischemic EAA baseline. Since
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Figure 2. Extracellular glutamate concentrations in dialysate
over the course of the experiment for sham (n54), normoglycemic (normo-) (n58), and hyperglycemic (hyper-) (n58) animals in
the hippocampal CA1 region and in the neocortex. Data are presented as mean6SEM. Fifteen minutes of ischemia (Isch) results
in an increase in extracellular glutamate concentration. This
increase was further enhanced in the cortex in rats loaded with
glucose. *P,0.05 vs baseline; †P,0.05 vs normoglycemic
group at identical times. B indicates baseline; R, minutes of
reperfusion.
there were no differences among them, the results of these 3
samples were pooled and served as baseline values.
Glutamate Concentrations
Preischemic extracellular glutamate baseline concentrations
in both the dorsal hippocampal region and the neocortex were
'20 pg/10 mL, with no differences among sham, normoglycemic, or hyperglycemic subjects.
The changes in extracellular glutamate concentrations in
the hippocampal CA1 subregion before, during, and after
ischemia are summarized in Figure 2, top panel. In shamoperated animals, the extracellular glutamate concentrations
were constant throughout the 180-minute sampling period. In
normoglycemic ischemic animals, extracellular glutamate
concentration increased from 20.362.2 to 284.8624.6 pg/10
mL (mean6SEM; 14.0-fold) after the induction of ischemia
(P,0.0001). The glutamate concentrations remained elevated
after 15 minutes of recirculation (150.8634.7 pg/10 mL;
P50.01) and returned to near-control levels thereafter.
Changes in glutamate of hyperglycemic rats were similar to
those recorded in normoglycemic ones. Thus, glutamate
content increased from 19.462.2 to 295.5672.3 pg/10 mL
during ischemia and remained elevated after 15 minutes of
recirculation (178.4642.6 pg/10 mL; P,0.01), subsequently
returning to baseline.
The glutamate transients were different in the neocortex
compared with the hippocampus. First, the elevation of
Figure 3. Extracellular glutamine concentrations in dialysate
(mean6SEM) over the course of the experiment for 3 groups in
the hippocampal CA1 region and in the neocortex. Glutamine
was decreased in normoglycemic animals, and the decrease
was more persistent in hyperglycemic animals. *P,0.05 vs
baseline. Abbreviations are as defined in Figure 2.
glutamate concentration during ischemia in normoglycemic
animals was less than that in the hippocampus (91.9626.1
versus 284.8624.6 pg/10 mL; P,0.01). Second, and most
importantly, hyperglycemic animals showed a more pronounced increase in glutamate content than normoglycemic
ones. As shown in Figure 2, bottom panel, glutamate levels
increased from 24.361.7 to 91.9626.1 pg/10 mL (3.8-fold;
P,0.01) in normoglycemic ischemic animals, whereas the
increase in glutamate was from 19.662.4 to 200.5639.8
pg/10 mL (10.2-fold; P,0.001) in hyperglycemic animals.
The peak value in hyperglycemic subjects was twice that in
normoglycemic animals (P,0.05).
Glutamine
Figure 3, top panel, illustrates changes in hippocampal
glutamine concentrations. Extracellular glutamine concentration decreased slightly in the normoglycemic group after the
induction of ischemia, and there was a further decrease at 15
and 30 minutes after recirculation (P,0.05). Afterward,
glutamine gradually recovered to baseline.
The baseline resting levels of glutamine were numerically
higher in the hyperglycemic group than in the normoglycemic
group. However, this difference was not statistically significant because of variations between animals. Compared with
normoglycemic animals, hyperglycemic animals had a more
prolonged and more long-lasting decrease in extracellular
glutamine during the ischemic and reperfusion periods. Thus,
extracellular glutamine contents remained lower than baseline during the 105 minutes of recirculation.
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Li et al
Figure 4. Extracellular glycine concentrations in dialysate
(mean6SEM) over the course of the experiment for 3 groups in
the hippocampal CA1 region and in the neocortex. The dialysate
glycine levels rose in both normoglycemic and hyperglycemic
animals. However, the increases were sustained longer in hyperglycemic than in normoglycemic groups. *P,0.05 vs baseline.
Abbreviations are as defined in Figure 2.
In the cortex, glutamine changes were not as pronounced as
in the hippocampus (Figure 3, bottom panel). Significant
decreases of glutamine concentration could only be detected
at 15 minutes of recirculation in normoglycemic animals
(P,0.05) and during the first 45 minutes of reperfusion in
hyperglycemic animals. However, the results reinforce the
impression that hyperglycemia prolongs the reduction in
glutamine concentration.
Glycine
The alterations in the extracellular glycine concentration in
the hippocampus are shown in Figure 4, top panel. Ischemia
induced an abrupt increase in extracellular glycine concentration in both normoglycemic and hyperglycemic groups.
Similar to the changes in glutamate, the peak concentrations
were reached after the induction of ischemia, and increased
levels were maintained during the immediate reperfusion
period. Unlike glutamate, however, the increase in glycine
was sustained in the postischemic period, and such a sustained increase lasted longer in hyperglycemic animals. In
normoglycemic animals the glycine concentration returned to
baseline level after 45 minutes of recirculation (P,0.05);
however, it was persistently increased (except in the 120minute sample) in hyperglycemic animals (P,0.05).
In the neocortex (Figure 4, bottom panel), glycine concentrations were moderately raised immediately after reperfusion
and declined to baseline values at 60 minutes of recovery in
the normoglycemic group. The increase in glycine concentration appeared somewhat delayed in the hyperglycemic rats,
Effect of Glucose on Excitatory Amino Acids
187
Figure 5. Extracellular taurine concentrations in dialysate
(mean6SEM) over the course of the experiment for 3 groups in
the hippocampal CA1 region and in the neocortex. The microdialysis taurine levels rose in response to the ischemic insult in
both normoglycemic and hyperglycemic animals. *P,0.05 vs
baseline; †P,0.05 vs normoglycemic group at identical times.
Abbreviations are as defined in Figure 2 legend.
but it was sustained for a longer period than in the normoglycemic animals.
Taurine
Figure 5 shows the alterations of extracellular taurine concentration in the hippocampal and cortical regions. Transient
increases of taurine in microdialysates were observed in both
normoglycemic and hyperglycemic subjects, and increased
values persisted for the first 30-minute period of recirculation
(P,0.01). The peak values appeared slightly lower in hyperglycemic than in normoglycemic animals.
Alanine
Extracellular alanine concentrations in the hippocampal CA1
subregion and cortex are presented in Figure 6. In the
hippocampus, alanine concentrations increased after ischemia
in the saline-infused rats. Unlike other amino acids, alanine
did not increase in concentration during ischemia and the
immediate reperfusion phase, but an increase occurred 45 to
90 minutes after reperfusion. There was no major change in
glucose-infused animals. Alanine changes in the cortex were
similar to those in the hippocampus.
Serine
Extracellular serine concentrations are given in Figure 7. In
both the cortex and the hippocampus, sham-operated animals
had a higher serine level than the 2 ischemic groups. Thus, the
neocortical values in hyperglycemic animals and hippocampal values in both normoglycemic and hyperglycemic groups
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Figure 6. Extracellular alanine concentrations in dialysate
(mean6SEM) over the course of the experiment for 3 groups in
the hippocampal CA1 region and in the neocortex. The microdialysis alanine levels rose slightly in response to the ischemic
insult in normoglycemic but not in hyperglycemic animals.
*P,0.05 vs baseline. Abbreviations are as defined in Figure 2.
were significantly lower than the values in the sham-operated
group. Extracellular serine concentrations were not changed
after ischemia and reperfusion in saline-infused rats. In
animals infused with glucose, a decrease in serine concentration could be observed in the hippocampus and the cortex
during ischemia. The decrease was also evident in samples
collected after 15 to 60 minutes of reperfusion.
Discussion
When the present results are discussed, it should be kept in
mind that even in normoglycemic animals, 15 minutes of
ischemia at a brain temperature of 37°C is a relatively severe
insult that, apart from giving close to 100% CA1 neuronal
necrosis, kills '30% of the neurons in the neocortical strip
examined. Damage to both of these structures is delayed, the
rate of evolution being faster in the neocortex than in the CA1
sector.15 It is thus possible to find reperfusion times when
neocortical but not CA1 damage is observed.
With this model, hyperglycemia results in such a severe
insult that survival is no longer possible, mainly because
intractable seizures develop.6,7 However, the ischemia does
not compromise reflow since the bioenergetic state recovers
upon reperfusion16 and capillary patency remains intact.17 It
is thus possible to use the first hour of reperfusion to study
events that prime the tissue for seizures and secondary
bioenergetic failure. In one respect, it would have been
advantageous to employ 10 minutes of ischemia and to use a
lower plasma glucose concentration since this would allow
hyperglycemic animals to survive.6,7 However, that model
Figure 7. Extracellular serine concentrations in dialysate
(mean6SEM) over the course of the experiment for 3 groups in
the hippocampal CA1 region and in the neocortex. The microdialysis serine levels rose in response to the ischemic insult in
both normoglycemic and hyperglycemic animals. *P,0.05 vs
baseline; †P,0.05 vs normoglycemic group at identical times.
Abbreviations are as defined in Figure 2.
carries the potential disadvantage of large interanimal
variations.
Another consideration is the difference in pHe (and pHi)
between the normoglycemic and hyperglycemic animals. As
measured under comparable ischemic conditions, the differences in pHe and pHi are '0.5 pH units, eg, pHe falls from 7.3
to 6.8 in normoglycemic animals and from 7.3 to 6.3 in
hyperglycemic animals.4,5 It is not known whether such a
difference could affect amino acid release during ischemia.
An additional consideration is the effect of anesthesia on
the measurements of EEAs. Equithesin contains magnesium,
which is a noncompetitive NMDA antagonist, and therefore it
would possibly have influenced the EEA levels. However,
since equithesin was used for implantation of cannulas 5 to 7
days before microdialysis samples were collected, it is
unlikely to have influenced the measurements of EEAs
conducted 5 to 7 days after the use of this anesthetic agent.
The rats that were kept under anesthesia for use in
microdialysis experiments showed no subclinical seizure
activity. Given the fact that the anesthesia obviously prevented seizure activity from occurring, it might influence the
measurements of EEAs. However, increases of extracellular
EEAs, including glutamate, were observed during ischemia
and in the immediate reperfusion period (15 minutes). It is not
likely that subclinical seizure activity develops at such an
early stage in reperfusion.18 In support, Nakashima and
Todd19 reported that anesthetic agents such as isoflurane and
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Li et al
pentobarbital do not influence the glutamate transient in rats
subjected to complete global ischemia.
Effect of Hyperglycemia on Brain Damage
Preischemic hyperglycemia worsens the ischemic injury (for
reviews, see References 2 and 3) Previous results have
demonstrated that while ischemia of 10 to 15 minutes’
duration results in maximal CA1 damage in normoglycemic
subjects, hyperglycemia aggravates damage in the parietal
cortex, striatum, and thalamus and recruits the cingulate
cortex, the CA3 sector of the hippocampus, and the substantia
nigra in the damage process. The present results confirm
these findings and provide an interesting observation of
extensive neocortical injury already present after 3 hours of
reperfusion. Predictably, damage to CA3 neurons was delayed to a later reperfusion period (see above). The mechanisms by which hyperglycemia exaggerates ischemic brain
damage have not been clarified. Acidosis may be one of the
key factors in hyperglycemia-related damage. This contention
is supported by the findings that acidosis induced by superimposed hypercapnia augments selective neuronal necrosis in
normoglycemic ischemic animals20 and in rats with hypoglycemic coma.21
Excitotoxic Theory and the Link Between Glucose
and Increased Glutamate
Numerous studies have shown that glutamate, when present
in excessive amounts extracellularly, is neurotoxic.8 –10,22,23
Glutamate and related EAAs activate postsynaptic glutamate
receptors. Activation of the a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA) type of glutamate receptor
allows influx of Na1, Cl2, and water (and a small amount of
Ca21) by way of receptor-gated channels. This leads to
swelling of the neurons.9 Activation of the N-methyl-Daspartate (NMDA) type of glutamate receptor opens a channel permeable to calcium, leading to intracellular calcium
overload. The resulting massive increase in intracellular
calcium has a number of consequences. The crucial event
may be the assembly of a mitochondrial permeability transition pore, which then triggers release of cytochrome c from
mitochondria, and a burst of production of reactive oxygen
species.24 –27
Our results demonstrate that the rise in neocortical extracellular glutamate concentration during ischemia and in the
immediate reperfusion period is more pronounced in hyperglycemic animals. Choi and colleagues28 previously tried to
assess the effect of hyperglycemia on extracellular glutamate
concentrations in rats subjected to brief periods of ischemia.
Their samples were taken from the hippocampus. This may
be why they did not observe an increased extracellular
glutamate concentration in hyperglycemic rats.
More than a decade ago, Gaitonde et al29 (1987) described
the pathway of glucose metabolism into EAAs. Approximately 70% of the 14C-labeled glutamate was formed from
acetyl coenzyme and 30% by 14CO2 fixation of pyruvate after
injection of [3,4-14C]glucose. Thus, glutamate in the brain is
formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. It
cannot be argued that hyperglycemia enhances glutamate
Effect of Glucose on Excitatory Amino Acids
189
synthesis. However, we cannot anticipate a relationship
between the total amount of glutamate formed and the
amount available for release during ischemia. Hyperglycemia
may also influence other factors determining the uptake of
amino acids. As will be discussed below, changes in glial
metabolism may explain some of the changes in glutamate
and glutamine concentrations.
Glial cells carry the main responsibility of rapid postischemic removal of EAAs accumulated in extracellular space.30
Increases in glutamate and decreases in glutamine could be
best explained by assuming that while some glial uptake of
glutamate may persist during ischemia, at least initially, the
subsequent metabolism of glutamate to glutamine is impeded.
The reduced metabolism of glutamate by glial cells may be
responsible for part of the extracellular glutamate overflow
during ischemia. However, an increased neuronal efflux of
glutamate is also likely to contribute.31,32 It seems likely that
postsynaptic neuronal elements as well as glial cells contribute to the extracellular overflow of EAAs during an ischemic
event.33
The additional increase of glutamate concentration in
hyperglycemic subjects may either reflect the fact that glucose enhances the formation and release of glutamate from
presynaptic endings, as described by Gaitonde et al29 (1987),
or reflect the fact that acidosis inhibits glutamate uptake by
glial cells (astrocytes).30 A reduction in pH from 7.4 to 5.8 in
primary rat astrocyte cultures causes a significant inhibition
of glutamate uptake, and this inhibition is more pronounced
when acidosis is combined with hypoxia.30 A similar situation
may have existed in our experiments, in which severe
acidosis is combined with cellular hypoxia.
It has been reported by Dietrich and colleagues34 (1993)
that hyperglycemia causes breakdown of the blood-brain
barrier after reperfusion following 20 minutes of global
ischemia, and it may therefore account for the increase in the
extracellular glutamate. However, since extracellular glutamate already increased during ischemia, it is unlikely that the
extra increase in extracellular glutamate resulted from the
breakdown of the blood-brain barrier.
It is not clear that an increased accumulation of glutamate
in extracellular fluid, above that observed in normoglycemic
animals, can explain the exacerbated cell damage in hyperglycemic subjects. However, since hyperglycemia-enhanced
glutamate release is correlated with aggravated damage observed in the neocortical areas, it is therefore possible that the
additional increase of glutamate concentrations in the extracellular space may be responsible, at least partially, for the
aggravated damage in hyperglycemic animals. It is not clear
why hyperglycemia enhances glutamate release in the neocortex but not in the hippocampus. Probably, maximal accumulation of glutamate in extracellular fluid already occurs in
normoglycemic rats; therefore, hyperglycemia cannot increase it further. It has been established that the hippocampus
has an abundance of glutamatergic inputs.35,36 This agrees
with our findings that the absolute values of extracellular
glutamate were much higher in the hippocampus than in the
neocortex. The failure of hyperglycemia to increase extracellular glutamate levels in the hippocampus is not compatible
with any general hypothesis of a coupling between glutamate
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190
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January 2000
concentration and hyperglycemia-exaggerated damage; in
fact, the data could be used to prove that such a coupling does
not exist. However, because of its vulnerability to ischemic
insults, the hippocampus could be a special case. Thus,
although the CA1 damage matures more rapidly in hyperglycemic subjects (present data and Reference 4), the ultimate
damage is already maximal in normoglycemic subjects. It
remains to be shown that a coupling exists between glutamate
release and hippocampal (CA1) damage when the duration of
ischemia is reduced, for example, to 5 minutes.
Changes in Glutamine
Our results demonstrate that a large increase in glutamate
efflux is coupled with a corresponding decline in glutamine
efflux. The decrease of glutamine is sustained for a longer
period in hyperglycemic animals. Glutamine is a major
metabolic precursor of glutamate. The reduction of extracellular glutamine may reflect utilization of intracellular glutamine for synthesis of glutamate or inhibition of synthesis
due to depletion of energy stores.37 However, the decrease in
extracellular glutamine could also be linked to an increased
activity of the phosphate-activated glutaminase, a key enzyme in the synthesis of neurotransmitter glutamate,38 since
tissue inorganic phosphate content increases markedly during
ischemia, and calcium promotes phosphate activation of
glutaminase. Inhibition of the glial enzyme glutamine synthetase, which converts glutamate to glutamine, has been
reported during reperfusion after cerebral ischemia.39 Suppression of activity of this enzyme could result in both a
reduction in glutamine efflux and synaptic glutamate
accumulation.
Prolonged Release of Glycine by Hyperglycemia
Glycine is located in presynaptic terminals. There are 2 types
of glycine receptors, Gly 1 (the classic strychnine-sensitive
inhibitory site) and Gly 2 (the strychnine-insensitive site).
Gly 1 acts as an inhibitory neurotransmitter because of its
ability to cause postsynaptic hyperpolarization. Gly 2 is
associated with excitatory transmission since it potentiates
NMDA activity. It has been assumed by some investigators
that glycine levels are probably already saturated under
physiological conditions and that changes in glycine are
unlikely to make any difference with respect to NMDA
receptor activation; however, many in vivo and in vitro
experiments provide convincing data that NMDA-associated
glycine sites are not saturated at physiological levels of
glycine (for review and literature, see Reference 40). Delayed
treatment with glycine site antagonists attenuates infarct size
in focal ischemia.41 In the present study glycine concentrations increased during ischemia, and the elevated levels were
sustained into the postischemic period. Such changes in
concentrations of glycine have been previously reported.28
The sustained increase may help to explain the ongoing
toxicity despite the return of glutamate to baseline levels. In
the present study, when compared with normoglycemic
animals, hyperglycemic animals had a much more prolonged
elevation in glycine levels in both hippocampal and neocortical areas after ischemia. It is tempting to postulate that the
rise in glycine modulates the action of glutamate in vivo
during and after ischemia and may be responsible, at least
partially, for the detrimental effect of hyperglycemia on
neurological outcome.
In summary, the present results demonstrate that preischemic hyperglycemia enhances the accumulation of extracellular glutamate in the neocortex and that the enhanced increase
of glutamate is correlated with exaggerated cell damage. The
increase was observed during ischemia and in the immediate
(15-minute) postischemic period. Hyperglycemia also prolonged the postischemic decrease in glutamine concentration
normally seen during the initial 15 to 30 minutes of recirculation and gave rise to a sustained increase in glycine
concentrations after recirculation. Changes in taurine, alanine, and serine concentrations were inconclusive. Thus,
although extracellular taurine and alanine concentrations rose
during ischemia and after reperfusion, no clear effects of
hyperglycemia were observed.
Acknowledgments
This study was supported by the Health Science Utilization and
Research Commission in Saskatchewan (Dr Li), the Canadian Heart
and Stroke Foundation (Dr Shuaib), and the Juvenile Diabetes
Foundation International (Dr Siesjö).
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Editorial Comment
It is clearly established that preischemic hyperglycemia
worsens ischemic outcome in laboratory animals. There also
is sufficient correlative human data for most clinicians to
accept the conclusion that glucose infusion should be withheld in patients at risk for ischemic brain damage. This
perhaps has greatest relevance to surgical patients, in whom
ischemic events can often be presaged, and it is also relevant
to patients undergoing cardiopulmonary resuscitation (CPR).
For these reasons, dextrose-containing solutions are usually
withheld from surgical patients, and the Advance Cardiac
Life Support algorithm has been modified to recommend
administration of normal saline as the principal intravenous
solution during CPR.1,2
From a scientific perspective, it remains a paradox that giving
glucose can worsen damage in ischemic tissue that is by
definition deprived of sufficient substrate to maintain normal
metabolic function. One likely mechanism for this is the enhanced lactic acidosis resulting from anaerobic metabolism of
glucose, which is disproportionately more available than oxygen. However, in vitro studies have found that acidosis actually
provides an advantage to cultured neurons deprived of metabolic
substrate.3 Accordingly, although lactic acidosis, in part, explains hyperglycemia-augmented ischemic brain damage, other
mechanisms are also likely involved.
It is widely accepted that disregulated glutamate metabolism during ischemia, and the concordant increase in extracellular glutamate concentration, contribute to ischemic brain
injury. It has been known that hyperglycemia augments
extracellular glutamate increases during ischemia.4 What is
novel in the current work by Li et al is the attempt to correlate
these changes with histological outcome. Rats were subjected
to severe forebrain ischemia while either normoglycemic or
hyperglycemic. Extracellular amino acid concentrations were
measured in both cortex and hippocampus. Data from the
cortex are probably most informative. As expected, extracellular glutamate increased during ischemia, and this increase
was greater in hyperglycemic rats. Corresponding to this,
cortical histological injury was greater in the hyperglycemic
group, which indicates a correlative relationship between
physiology and outcome.
Data from the hippocampus are more difficult to interpret.
Again, ischemia increased glutamate concentrations. How-
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ever, because hyperglycemic animals typically died within
the first few hours after ischemia, histological information
could not be obtained when full maturation of hippocampal
injury would be expected to occur. Because normoglycemic
rats subjected to the same insult survived 7 days and were
found to have 100% hippocampal CA1 damage, it is likely
that the ischemic insult alone was sufficient to cause maximal
glutamate accumulation, disallowing any further augmentation by hyperglycemia. This could be confirmed in future
studies by repeating the experimental protocol with a shorter
duration of ischemia, which would result in only moderate
damage in the CA1 sector in normoglycemic rats.
The results of this work by Li et al are consistent with the
hypothesis that hyperglycemia worsens ischemic outcome, at
least in part, by augmenting extracellular glutamate accumulation, which in turn upregulates N-methyl-D-aspartate receptor activation and conductivity to calcium. However, data to
date remain only correlative. The importance of this limitation can be demonstrated by examining effects of hypothermia on glutamate concentrations during ischemia. It is known
that hypothermia nearly abolishes any increase in glutamate
concentration.5 However, recent evidence provided by
Yamamoto et al6 questions the importance of this effect.
When hypothermic gerbils underwent microinjection of glutamate into hippocampal CA1 in sufficient quantity to cause
intraischemic extracellular glutamate concentrations to be
similar to those observed in normothermic gerbils, hypothermia still caused a profound reduction in histological damage.
Accordingly, additional work is required to confirm a causal
relationship between hyperglycemia and glutamatergic excitotoxicity. Because NMDA receptor activation is associated
with increased permeability of calcium, assessment of calcium transients in normoglycemic versus hyperglycemic
animals would be important. Also, because glutamate excitotoxicity has been linked to opening of the mitochondrial
permeability transition pore,7 investigation of mitochondrial
responses to hyperglycemic versus normoglycemic ischemia
should be examined. The results of Li et al justify further
pursuit of these questions.
David S. Warner, MD, Guest Editor
Department of Anesthesiology
Duke University Medical Center
Durham, North Carolina
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